Summary Identifying the spatial organization of tissues at cellular resolution from single cell gene expression profiles is essential to understanding neurological systems. We have developed a spatial genomics approach that allows in situ 3D multiplexed imaging of many genes in single cells called sequential Fluorescence in situ hybridization (seqFISH). This technology can profile transcriptional states of single cells directly in their native tissue context with up to 249 genes multiplexed with single molecule sensitivity on each gene. We have demonstrated over 15,000 cells profiled in mouse brain slices. This SBIR project will be focused on the design, production and optimization of an instrument that allows hundreds of genes to be multiplexed and imaged in single cells within their native tissue context. The resulting machine will be commercially launched and targeted to imaging or sequencing cores at research institutions. We will design the hardware, code the control software, and build the prototype instrument. We will engineer the hardware component including automated fluidics and multiple camera imaging system with a parallel effort to develop software controls as well as integrated analysis tools. In phase II, we will beta-test the instrument, generate probe sets for gene panels targeting different brain samples, and receive valuable feedback from users and optimize our instrument design.